Bottom Line:
Unexpectedly, neither suppressor mutation corrected the intrinsic viral capsid stability defect associated with the respective original mutation.We propose that while proper HIV-1 uncoating in target cells is dependent on the intrinsic stability of the viral capsid, the effects of stability-altering mutations can be mitigated by additional mutations that affect interactions with host factors in target cells or the consequences of these interactions.The ability of mutations at other CA surfaces to compensate for effects at the NTD-NTD interface further indicates that uncoating in target cells is controlled by multiple intersubunit interfaces in the viral capsid.

Affiliation: Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN, USA.

ABSTRACT

Background: Disassembly of the viral capsid following penetration into the cytoplasm, or uncoating, is a poorly understood stage of retrovirus infection. Based on previous studies of HIV-1 CA mutants exhibiting altered capsid stability, we concluded that formation of a capsid of optimal intrinsic stability is crucial for HIV-1 infection.

Results: To further examine the connection between HIV-1 capsid stability and infectivity, we isolated second-site suppressors of HIV-1 mutants exhibiting unstable (P38A) or hyperstable (E45A) capsids. We identified the respective suppressor mutations, T216I and R132T, which restored virus replication in a human T cell line and markedly enhanced the fitness of the original mutants as revealed in single-cycle infection assays. Analysis of the corresponding purified N-terminal domain CA proteins by NMR spectroscopy demonstrated that the E45A and R132T mutations induced structural changes that are localized to the regions of the mutations, while the P38A mutation resulted in changes extending to neighboring regions in space. Unexpectedly, neither suppressor mutation corrected the intrinsic viral capsid stability defect associated with the respective original mutation. Nonetheless, the R132T mutation rescued the selective infectivity impairment exhibited by the E45A mutant in aphidicolin-arrested cells, and the double mutant regained sensitivity to the small molecule inhibitor PF74. The T216I mutation rescued the impaired ability of the P38A mutant virus to abrogate restriction by TRIMCyp and TRIM5α.

Conclusions: The second-site suppressor mutations in CA that we have identified rescue virus infection without correcting the intrinsic capsid stability defects associated with the P38A and E45A mutations. The suppressors also restored wild type virus function in several cell-based assays. We propose that while proper HIV-1 uncoating in target cells is dependent on the intrinsic stability of the viral capsid, the effects of stability-altering mutations can be mitigated by additional mutations that affect interactions with host factors in target cells or the consequences of these interactions. The ability of mutations at other CA surfaces to compensate for effects at the NTD-NTD interface further indicates that uncoating in target cells is controlled by multiple intersubunit interfaces in the viral capsid.

Figure 2: Rescue of P38A and E45A infectivity defects mutations by second-site mutations T216I and R132T. Single-cycle infectivity was assayed in HeLa-P4 target cells. Infectivity was determined as the number of infected cells per ng of p24 in the inoculum, and values are expressed as percentage of wild-type HIV-1 infectivity. Results shown are the mean values of three independent experiments, with error bars representing one standard deviation.

Mentions:
Our group previously showed that the P38A and E45A mutants are impaired at an early post-entry stage of infection [17]. Therefore, we sought to determine whether the second-site mutations rescue the P38A and E45A mutant viruses in a single-round reporter assay. This assay, which requires expression of Tat from an integrated provirus, is a useful tool to identify defects in the early stages of infection. Quantitation of virus infectivity using the HeLa-P4 cell line (HeLa-CD4/LTR-lacZ) revealed that the T216I and R132T second-site mutations markedly enhanced the infectivity of P38A and E45A, respectively (Figure 2). We also constructed and determined the infectivity of the single mutants T216I and R132T. Both mutants exhibited infectivity similar to the wild type virus (data not shown). Therefore, the second-site mutations function by relieving a defect in a step of the virus infection cycle prior to integration.

Figure 2: Rescue of P38A and E45A infectivity defects mutations by second-site mutations T216I and R132T. Single-cycle infectivity was assayed in HeLa-P4 target cells. Infectivity was determined as the number of infected cells per ng of p24 in the inoculum, and values are expressed as percentage of wild-type HIV-1 infectivity. Results shown are the mean values of three independent experiments, with error bars representing one standard deviation.

Mentions:
Our group previously showed that the P38A and E45A mutants are impaired at an early post-entry stage of infection [17]. Therefore, we sought to determine whether the second-site mutations rescue the P38A and E45A mutant viruses in a single-round reporter assay. This assay, which requires expression of Tat from an integrated provirus, is a useful tool to identify defects in the early stages of infection. Quantitation of virus infectivity using the HeLa-P4 cell line (HeLa-CD4/LTR-lacZ) revealed that the T216I and R132T second-site mutations markedly enhanced the infectivity of P38A and E45A, respectively (Figure 2). We also constructed and determined the infectivity of the single mutants T216I and R132T. Both mutants exhibited infectivity similar to the wild type virus (data not shown). Therefore, the second-site mutations function by relieving a defect in a step of the virus infection cycle prior to integration.

Bottom Line:
Unexpectedly, neither suppressor mutation corrected the intrinsic viral capsid stability defect associated with the respective original mutation.We propose that while proper HIV-1 uncoating in target cells is dependent on the intrinsic stability of the viral capsid, the effects of stability-altering mutations can be mitigated by additional mutations that affect interactions with host factors in target cells or the consequences of these interactions.The ability of mutations at other CA surfaces to compensate for effects at the NTD-NTD interface further indicates that uncoating in target cells is controlled by multiple intersubunit interfaces in the viral capsid.

Affiliation:
Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN, USA.

ABSTRACT

Background: Disassembly of the viral capsid following penetration into the cytoplasm, or uncoating, is a poorly understood stage of retrovirus infection. Based on previous studies of HIV-1 CA mutants exhibiting altered capsid stability, we concluded that formation of a capsid of optimal intrinsic stability is crucial for HIV-1 infection.

Results: To further examine the connection between HIV-1 capsid stability and infectivity, we isolated second-site suppressors of HIV-1 mutants exhibiting unstable (P38A) or hyperstable (E45A) capsids. We identified the respective suppressor mutations, T216I and R132T, which restored virus replication in a human T cell line and markedly enhanced the fitness of the original mutants as revealed in single-cycle infection assays. Analysis of the corresponding purified N-terminal domain CA proteins by NMR spectroscopy demonstrated that the E45A and R132T mutations induced structural changes that are localized to the regions of the mutations, while the P38A mutation resulted in changes extending to neighboring regions in space. Unexpectedly, neither suppressor mutation corrected the intrinsic viral capsid stability defect associated with the respective original mutation. Nonetheless, the R132T mutation rescued the selective infectivity impairment exhibited by the E45A mutant in aphidicolin-arrested cells, and the double mutant regained sensitivity to the small molecule inhibitor PF74. The T216I mutation rescued the impaired ability of the P38A mutant virus to abrogate restriction by TRIMCyp and TRIM5α.

Conclusions: The second-site suppressor mutations in CA that we have identified rescue virus infection without correcting the intrinsic capsid stability defects associated with the P38A and E45A mutations. The suppressors also restored wild type virus function in several cell-based assays. We propose that while proper HIV-1 uncoating in target cells is dependent on the intrinsic stability of the viral capsid, the effects of stability-altering mutations can be mitigated by additional mutations that affect interactions with host factors in target cells or the consequences of these interactions. The ability of mutations at other CA surfaces to compensate for effects at the NTD-NTD interface further indicates that uncoating in target cells is controlled by multiple intersubunit interfaces in the viral capsid.